Epidermal Growth Factor (EGF) regulates a variety of biological events ranging from cell growth and proliferation to intermediary metabolism, heart rate and contractility. In the heart, EGF increases beating rate and contractility by elevating cAMP levels. EGF-mediated increase in cAMP levels in the heart is the result of stimulation of adenylyl cyclase (AC) via activation of the stimulatory GTP binding protein Gs. A cytosolic, juxtamembrane, 13 amino acid sequence of the EGF receptor (EGFR) can activate Gs. Moreover, phosphorylation of Gsalpha by the EGFR can also activate the G protein. Since the association of Gsalpha with the juxtamembrane 13 amino acid region of EGFR is necessary for the efficient phosphorylation of the G protein, studies in aim 1 will pursue experiments to identify critical amino acids in the juxtamembrane region of the EGFR which are necessary for association of Gsalpha and activation of the G protein. Recently, we have shown that in a number of cell types EGF does not increase cAMP levels. However, in these cells the cAMP-dependent protein kinase (PKAc) activity is increased in response to EGF. Moreover, using purified PKAc and EGFR we have demonstrated that the EGFR can stoichiometrically phosphorylate PKAc and activate this protein. Therefore, studies in aim 2 will elucidate the mechanism(s) involved in cAMP-independent activation of PKAc by the activated EGFR in-vitro as well as in intact cells. Moreover, we have shown that PKAc can stoichiometrically phosphorylate the purified EGFR on Ser residues and attenuate the protein tyrosine kinase activity of the EGFR. Exposure of cells to the cAMP analog 8CPT-cAMP also attenuates EGF-mediated tyrosine phosphorylation of cellular proteins and EGFR autophosphorylation. Hence, under aim 3, we will identify the Ser residues which are phosphorylated by PKAc and determine whether phosphorylation of these residues is necessary and sufficient to inhibit EGFR tyrosine kinase activity. We will also perform experiments to determine if PKAc activation by the EGF in cells serves as a feed-back regulatory mechanism in EGF-elicited activation of AC. Overall, our experiments will delineate the mechanisms involved in interactions between EGFR and PKA signaling pathways. Since both of these pathways manifest numerous biological actions, our findings will also begin to explain how activation of one of these pathways regulates events triggered by the other.